噬菌体制剂雾化治疗多重耐药肺炎克雷伯菌感染小鼠的效果评价  

Evaluation of Therapeutic Efficacy of Multi-drug Resistant Klebsiella pneumoniae in a Mouse Model by Inhalation of Nebulized Bacteriophage

作　　者：沈秀平 张释丹 刘源平 曾小艳 王兆飞 SHEN Xiuping;ZHANG Shidan;LIU Yuanping;ZENG Xiaoyan;WANG Zhaofei(Shanghai Agricultural Product Quality and Safety Center,Shanghai 201708,China;Shanghai Key Laboratory of Veterinary Biotechnology,School of Agriculture and Biology,Shanghai Jiao Tong University,Shanghai 200240,China)

机构地区：[1]上海市农产品质量安全中心,上海201708 [2]上海市兽医生物技术重点实验室、上海交通大学农业与生物学院,上海200240

出　　处：《中国生物工程杂志》2025年第2期1-12,共12页China Biotechnology

基　　金：上海市农业科技创新(2022-02-08-00-12-F01182)资助项目。

摘　　要：目的:肺炎克雷伯菌是临床常见的多重耐药菌,急需寻找新型治疗制剂。借助噬菌体的特异性杀菌作用,构建小鼠多重耐药肺炎克雷伯菌肺炎模型并评估噬菌体雾化治疗效果。方法:从医院污水中分离获得肺炎克雷伯菌噬菌体,通过测定噬菌体一步生长曲线、裂菌谱和全基因组测序,评估噬菌体作为候选噬菌体抗菌制剂的杀菌效力。使用高毒力耐药肺炎克雷伯菌进行小鼠滴鼻攻毒,将纯化的噬菌体液进行小鼠雾化治疗。检测小鼠雾化治疗后肺组织中的细菌载量、噬菌体效价和免疫相关基因表达水平,比较肺组织病理切片的变化,使用荧光标记的白蛋白评估小鼠肺部屏障功能的完整性。结果:分离获得一株肺炎克雷伯菌噬菌体vB_KpnP_SY201(SY201),属于短尾噬菌体科。一步生长曲线表明其感染20 min后即可进入裂解期,并可高效裂解高毒力耐药肺炎克雷伯菌HG-KP-3,具有作为治疗制剂的应用潜力。同时,全基因组测序结果验证其无毒力基因或整合酶位点,具备治疗的安全性。在小鼠急性肺炎治疗中,噬菌体雾化治疗相较于滴鼻治疗能在肺部聚集更多的噬菌体。噬菌体雾化治疗可使肺部细菌载量减少至未治疗组的1/30。肺组织病理切片和荧光标记白蛋白观察结果表明,雾化治疗组的小鼠肺组织结构基本恢复,未见炎性浸润和病灶,屏障功能恢复,且免疫相关基因IL-6、IL-1β和TNF-α的表达下降。以上结果均提示噬菌体雾化吸入方法可有效治疗小鼠肺炎。结论:噬菌体制剂雾化治疗方法具有良好的临床应用潜力,是治疗耐药性细菌肺部感染的潜在优质策略。Objective:Effective therapeutic agents against multidrug-resistant Klebsiella pneumoniae(K.pneumoniae)are urgently needed in clinical practice.We developed a mouse model of pneumonia induced by multidrug-resistant K.pneumoniae to evaluate the efficacy of phage nebulization as a treatment.Methods:A K.pneumoniae-specific phage was isolated from hospital wastewater.The phage one-step growth curve,host spectrum,and complete genome sequencing were used to evaluate the bactericidal efficacy of the phage as a potential antimicrobial agent.Mice were challenged by nasal inhalation with highly virulent and multi-drug resistant K.pneumoniae.Purified phage was obtained by density gradient centrifugation for nebulization therapy in mice.After nebulization,mouse lung tissu was examined for bacterial load,phage titers,and immune-related gene expression levels.Additionally,changes in histopathologic sections of the lungs were compared,and fluorescence-labeled albumin was used to evaluate the integrity of the lung barrier function in mice.Results:We isolated a K.pneumoniae phage,SY201,which belongs to the Podoviridae family.The phage entered the lysis phase within 20 mins of infection and effectively lysed the highly virulent,drug-resistant K.pneumoniae strain HG-KP-3,suggesting its potential as a therapeutic agent.Whole genome sequencing confirmed the phage’s safety profile,showing no virulence genes or integrase loci.Nebulization therapy significantly reduced lung bacterial load by approximately 30-fold compared to nasal drip therapy.Histopathologic analysis indicated restored lung tissue structure and barrier function,with no inflammation or lesions observed.Expression levels of immune-related genes IL-6,IL-1βand TNF-αdecreased,indicating effective treatment of pneumonia in mice.Conclusion:The combination of phage therapy and nebulization technique holds promise for clinical application,and offers a superior treatment option for pulmonary infections.

关 键 词：肺炎克雷伯菌 多重耐药 小鼠肺炎模型 噬菌体 雾化治疗 

分 类 号：Q819[生物学—生物工程]